Droplet-based assays, in which micro-scale emulsions are used as isolated compartments to run many independent chemical reactions, have gained popularity in recent years as a platform for a wide range of biomedical applications. Compared to the conventional laboratory approach of using millimeter-sized well plates to isolate fluids, micrometer-scale droplets contain only picoliters (10-12 L) of fluid, offering a 106× reduction in volume. Furthermore, compared to the hundreds of compartments available on a conventional well plate, microfluidics allow droplets to be created at rates as high as 106 per minute, offering a greater than 104 times increase in the number of compartments over conventional techniques. The enormous increase in sensitivity that comes from massively parallelized, ultra-small volume assays, has been harnessed to detect both single molecules of protein and nucleic acid, to monitor molecular concentrations as a function of time, to perform high-throughput screens for directed evolution, and to assay single cells.
While the microfluidics to produce and process droplets can be miniaturized and integrated onto compact, monolithic chips, the read-out of droplet-based assays have been more difficult to miniaturize. Fluorescence-based sensing may be used because: 1) molecular beacons, which can turn on or off fluorescence based on binding events, obviate extra steps to wash away excess reagents; 2) differently colored fluorophores allow for the detection of multiple targets in a single droplet; and 3) widely available fluorescence-based reagents ease assay development. Previous work has been done to integrate fluorescence detection with droplet microfluidics and to miniaturize fluorescence detection of cells. Wide-field microscopy techniques have been developed that can take micrographs of static droplets, with an ability to resolve as many as 106 in a single-shot. Other groups have developed in-flow detection systems, which have the advantage of real-time sorting, down-stream processing, and an ability to measure a far greater number of droplets than possible with the static techniques, measuring as many as 104 droplets per second. However, these techniques require complex optics and are not easily amenable to monitoring more than one channel.